Nucleic acid quantitation from tissue slides

ABSTRACT

This invention provides methods of quantitating nucleic acids from problematic samples, such as aged samples, formalin fixed samples, paraffin embedded samples, samples with aneuploid cells, and cells with fragmented nucleic acids. Methods include techniques to efficiently solubilize the nucleic acids under non-denaturing conditions from preserved clinical samples without resort to organic extractions, to normalize cell counts regardless of aneuploidy, to access the fragmentation state of the nucleic acids, and to provide standard curves for degraded nucleic acid samples.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and benefit of a prior U.S.Provisional Application No. 60/838,578, Nucleic Acid Quantitation fromTissue Slides, by Gary McMaster, et al., filed Aug. 17, 2006. The fulldisclosure of the prior application is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention is in the field of nucleic acid extraction andquantitation from cells and tissues. Nucleic acids are extracted fromembedded clinical samples without the use of hydrophobic solvents.Ribosomal DNA references and/or degraded in vitro RNAs are used tonormalize standard curves and to establish nucleic acid copy numbers percell.

BACKGROUND OF THE INVENTION

Formalin-fixed, paraffin-embedded (FFPE) tissue offers a vast source ofbiopsy specimens for which the clinical outcome is well documented andthus an optimal resource for retrospective studies (Lewis, F. et al2001; Yang et al 2006). Access to and use of human specimens is anessential part of the cancer research and drug discovery infrastructure,enabling researchers to identify drug targets, develop lead compoundsand understand drug metabolism. Research using human specimens can helppredict drug response and toxicity, as well as short and long termclinical outcome. New technologies and information gained from mappingthe human genome continue to fuel a growing need for researchers inacademia and industry; for-profit and not-for-profit, to have access togood quality human specimens to expedite cancer drug discovery. Manydifferent types of human specimens are required to support thesestudies: normal and malignant tissues, blood, other fluids, and theproteins, DNA and RNA that can be extracted from them.

Because all surgical procedures performed in the U.S. must obtain tissuesamples for pathology diagnosis, primary sources for human specimens arehospital operating rooms and pathology laboratories. More than 160million pathology specimens (most of them fixed tissue in wax blocks)are currently stored in the United States (Eiseman and Haga, 1999).

Tissue blocks are routinely fixed and embedded in paraffin, thensectioned with a microtome, and the sections affixed to microscopeslides. The paraffin-embedded tissue sections have required dewaxingprior to analysis of nucleic acids to allow penetration by aqueoussolutions.

For example, using a clean razor blade, FFPE sections have been scrapedoff slides and transferred into microfuge tubes for processing. Thetraditional method of paraffin removal involves organic extractionsusing xylene and graded alcohols. This procedure is time-consuming,cumbersome, and requires special handling, as xylene is a highly toxicchemical that emits noxious fumes. After 10 sections (60-100microns/25-250 mm²) of FFPE specimens from the same tissue block arescraped off from glass slides into the tubes using a scalpel, onemilliliter of hydrophobic solvent is added, e.g., xylene-containingEZDeWax™ (BioGenex, San Ramon, Calif., USA); see FIG. 1. After vortexmixing and incubating at room temperature for 5 min, the tissue samplesare centrifuged in a microcentrifuge at 16,000×g for 2 min, and thesupernatants removed. One milliliter 70% ethanol can be added to thesamples, and the samples vortex mixed and centrifuged in amicrocentrifuge at 16,000×g for 2 min. The sample wax is then extractedrepeatedly into the xylene phase and the residue washed with 70% ethanolfor two-five more times before continuing to the next step of tissuehomogenate preparation or total RNA isolation (Yang et al., 2006).

The phase extraction dewaxing protocols are time consuming andlaborious. The repeated handling, aspirations and tube transfers canresult in non-quantitative harvests of the nucleic acids. The repeatedvortexing and exposure to harsh solvents can cause sample degradation.

Additional problems exist in the quantitation of nucleic acids frompreserved clinical specimens. For example, RNA quality can be affectedby sample collection, formalin fixation and tissue processing. This cancompromise, e.g., the ability to measure RNA in FFPE tissue blocks. Thenucleic acids ultimately extracted from embedded clinical samples areoften highly degraded and fragmented. Qualitative and quantitative assayerrors often result when these extracts are evaluated by standardanalytical techniques. What's more, incomplete extractions can introduceerror into calculations, such as mRNA copy number determinations. Oneproblem in measuring RNA from FFPE tissue blocks can be fragmentation ofthe RNA fragments, cross-linking, and base modifications induced byformalin-fixation procedures. Two processes that reduce the length ofRNA molecules in formalin-fixed tissues are degradation andfragmentation (hydrolysis). RNA degradation can occur through enzymaticcleavage before the tissue encounters a fixative and is thus subject tothe collection procedure of the samples. Fragmentation of RNA moleculescan be caused by the formalin fixative and therefore variessubstantially depending on formalin conditions employed (Lehmann U,Kreipe H: Real-time PCR analysis of DNA and RNA extracted fromformalin-fixed and paraffin-embedded biopsies, Methods 2001,25:409-418). The exact causes for the fragmentation are not known, andthus it has been unclear how to solve this problem.

The current state-of-the-art technology for measuring RNA isquantitative PCR (QPCR). However, several recent reports comparing RNAquantification in frozen and FFPE tissues demonstrate that only 3-5% ofRNA transcripts are available for detection by QPCR after formalinfixation (Bibikova M, Talantov D, Chudin E, Yeakley J M, Chen J, DoucetD, Wickham E, Atkins D, Barker D, Chee M, Wang Y, Fan J B: Quantitativegene expression profiling in formalin-fixed, paraffin-embedded tissuesusing universal bead arrays, Am J Pathol 2004, 165:1799-1807). Thisproblem is independent of whether the reverse transcription step usesoligo-dT or random priming. A viable explanation for this problem isthat reverse transcription and/or QPCR are severely affected by formalinmediated mono-methylolation of bases in RNA. Attempting to compensatefor this problem, the expression of genes of interest has beennormalized to internal housekeeping genes. However, this is ofteninadequate because adenines are more susceptible to alteration byformalin fixation and thus A/U rich sequences will be less accuratelymeasured than G/C rich sequences (Masuda N, Ohnishi T, Kawamoto S,Monden M, Okubo. K: Analysis of chemical modification of RNA fromformalin-fixed samples and optimization of molecular biologyapplications for such samples, Nucleic Acids Res 1999, 27:4436-4443).Consequently, there will be gene specific differences in the efficiencyand reproducibility of measuring mRNA in formalin-fixed tissues. Inaddition to its modification by formalin, the heavy fragmentation duringthe fixation and/or subsequent isolation process requires specializedprimer design. Thus, there are severe limitations for PCR-based RNAmeasurements in formalin-fixed tissues. Alternative methods that areless sensitive to formalin-induced alterations are needed to improve theaccuracy of RNA quantification (Bustin S A: Quantification of mRNA usingreal-time reverse transcription PCR (RT-PCR): trends and problems, J MolEndocrinol 2002, 29:23-39; Bustin S A, Nolan T: Pitfalls of quantitativereal-time reverse-transcription polymerase chain reaction, J Biomol Tech2004, 15:155-166 Gunther E C, Stone D J, Gerwien R W, Bento P, Heyes MP: Prediction of clinical drug efficacy by classification ofdrug-induced genomic expression profiles in vitro, Proc Natl Acad SciUSA 2003, 100:9608-9613):

Performance of quantitative PCR (QPCR) has faired poorly in quantitationof FFPE RNAs because it is generally limited to 75-85 bp amplicon size,and multiple pooled gene-specific primers are required (Cronin M, Pho M,Dutta D, Stephans J C, Shak S, Kiefer M C, Esteban J M, Baker J B:Measurement of gene expression in archival paraffin-embedded tissues:development and performance of a 92-gene reversetranscriptase-polymerase chain reaction assay, Am J Pathol 2004,164:35-42). QPCR requires a much greater purity of RNA than the bDNAassay and thus more steps to process the samples prior to analysiscompared to the bDNA technology. After dewaxing, the RNA needs to bedigested with Proteinase K, isolated and submitted to 1-2 times ofDNAase I treatment to remove DNA contamination. A second problem thataffects. RNA quantification by QPCR is the required reversetranscription step to convert mRNA sequences of interest to cDNA. Thisenzymatic reaction is impeded by formalin-induced base modifications, bysecondary mRNA structure and by impurities in the RNA preparation.Factors inhibiting reverse transcription will vary amongst FFPE tissueblocks. Although, introduction of a high temperature heating step duringPCR amplification steps may partially reverse some of the RNA basemodifications, for many samples these modifications are irreversible.Older samples are often so impaired that a decrease in average QPCRsignal is >90%, requiring more input RNA and increasing Ct values to35-40 (Masuda N, Ohnishi T, Kawamoto S, Monden M, Okubo K: Analysis ofchemical modification of RNA from formalin-fixed samples andoptimization of molecular biology applications for such samples, NucleicAcids Res 1999, 27:4436-4443). With all these problems, QPCR has notbeen a satisfactory method of quantitating RNAs from FFPE samples.

In view of the above, a need exists for a faster and simpler way toharvest nucleic acids from embedded clinical tissue samples. It would bedesirable to have a way to obtain nucleic acids from formalin fixedparaffin embedded samples without the use of hazardous solvents. Theaccuracy of nucleic acid analyses would benefit from a more quantitativeand less damaging methods of nucleic acid extraction. Benefits can beobtained from methods to adjust analyses to take target degradation intoconsideration. The present invention provides these and other featuresthat will be, apparent upon review of the following.

SUMMARY OF THE INVENTION

Methods of the invention are useful in addressing problems encounteredin analysis of nucleic acid samples that are physically difficult toprocess or have experienced degradation. The methods can help obtaingood representative test materials from samples that were previouslyprocessed for evaluation of histopathology. The methods can increase theaccuracy and sensitivity of sample analyses by providing morerepresentative standard materials. The condition of possibly degradednucleic acids can be determined by an inventive offset bDNA: assayconfigured for increased sensitivity to target fragmentation. Themethods can improve estimates of mRNA copy counts for test materialsderived from unknown numbers of normal and/or abnormal Cells. Thetechniques can be used in combination to optimize nucleic acid analysesof, e.g., test materials derived from normal and/or aneuploid cells,formalin fixed cells, paraffin embedded cells, aged clinical samples,and the like.

Methods of the invention include combinations of inventive techniquesworking together to enhance the sensitivity and accuracy of a nucleicacid determination. For example, mRNA copy numbers can be estimatedaccurately by determining the number of cells in a test sample (e.g., bycomparing a test sample rDNA value to a standard function of rDNA versuscell number); preparing a standard function for an RNA assay (e.g.,assay output versus a degraded in vitro RNA standard assayinput-degradation of sample or standard determined, e.g., by an offsetbDNA assay), determining an amount of a test mRNA in the test sampleusing the RNA assay standard function, and determining the copy numberof the mRNA in the test cells based on the number of cells and thedetermined amount of test mRNA. This procedure can effectively determinemRNA copy numbers in a variety of cell and tissue types, such as, e.g.,tumor cells, cell lines, cells from a microscope slide, clinical samplesmore than a year old, fresh tissue, freshly fixed cells, freshly fixedparaffin embedded tissues, cells fixed with formalin, cells embedded inparaffin, normal and/or aneuploid cells, and the like e.g., originatingfrom humans, plants, or animals.

In one embodiment of the inventive techniques, a fast, simple,quantitative and reliable technique is provided to release nucleic acidsfrom samples, such as formalin fixed paraffin embedded (FFPE) clinicalsamples, embedded in a matrix of hydrophobic media. In a general aspect,a method of collecting a nucleic acid from cells associated with ahydrophobic component can include suspending the sample, incubating thesample and separating nucleic acids from the sample and hydrophobiccomponent. The sample of cells or tissue with the hydrophobic componentmelting at a temperature greater than 40° C. can be suspended in anaqueous solution. The suspension can be incubated at a temperaturehigher than 40° C. under conditions substantially non-denaturing todouble stranded DNA of the cells, so that the hydrophobic componentmelts and the nucleic acid is released from the cells into the aqueoussolution. Finally, the aqueous solution can be physically separated fromthe hydrophobic component, after the incubation, to collect the nucleicacid released from the Cells.

This method of nucleic acid release or solubilization can work well formany cell and/or tissue samples. For example, the methods can be used toprepare aqueous test materials useful in analyses of DNA, a degradednucleic acid, RNA, and the like. The methods are particularly useful toprovide test samples for nucleic acid analysis of clinical samplescontaining a wax such as formalin fixed paraffin embedded tissue orcells.

Suspending the cells or tissue in the aqueous solution can be by anappropriate technique. For example, a tissue sample on a microscopeslide can be scrapped off into an Eppendorf tube and vortexed. Thickeror more stubborn samples can be broken into smaller particles, e.g., bygrinding, chopping, pressing, douncing, milling, and the like. Theaqueous solution can include constituents designed to help disrupt thecells and tissues, to aid in the solubilization of the nucleic acids,and/or to condition the solution for the intended analysis. For example,the aqueous solution (water containing a solute) can include PEG, SDS,SSC buffer, NaHPO4, EDTA, denatured salmon sperm DNA, divalent cations,formamide, SSPE buffer, blocking probes, capture extenders, labelextenders, preamplifiers, label probes, amplification probes,amplification multimers, a protease, a lipase, a surfactant, or nucleaseinhibitor, and/or the like. In a preferred embodiment, the aqueoussolution optionally contains a protease, such as proteinase K, at 10ul/ml, 50 ul/ml, 100 ul/ml, 150 ul/ml, 250 ul/ml, 500 ul/ml, 1 mg/ml, ormore.

Incubation in the method is for a time and temperature suitable torelease the desired nucleic acid from the sample in an amount andconcentration adequate for the intended analysis. Using associatedmethods of the invention, complete release of all nucleic acids from thesample is often not required because analyses can be standardized andnormalized to provide meaningful results. In typical embodiments, theincubation is carried out at a temperature ranging from about 35° C. toabout 99° C., from about 45° C. to about 95° C., from about 52° C. toabout 90° C., from about 60° C. to less than 80° C., or about 65° C.Preferably, the incubation temperature is above the melting point of apredominant sample hydrophobic component by at least a couple ofdegrees, but below the Tm of the sample DNA under the conditions of thesuspension. Incubation can be rapid, particularly at higher temperaturesor for delicate or fine samples. Incubation time can be more than 20minutes, or range from about 30 minutes to about 3 days or more, fromabout 1 hour to 1 day, from about 3 hours to about 18 hours, or 12hours: In preferred embodiments, the incubation can be started in theafternoon and proceed over night for analysis in the morning. Methods ofthe invention allow a certain lack of precision in many sample handlingsteps due to the ability of the methods to correct for handlingvariables.

In many embodiments, the aqueous solution and incubation conditions donot include nucleic acid denaturing conditions, e.g., conditions thatwould melt most of the sample DNA from double stranded form to singlestranded form. Denaturing conditions, as well known in the art, caninclude increased solution temperature, high pH, and high ionicstrength.

Separating the hydrophobic component from the aqueous solution orsuspension can be, e.g., by simple mechanical (e.g., solely physical)means. Although it has been the practice to separate paraffin from FFPEsamples using chemical extractions (e.g., organic phase extractions), wefind physical separation (e.g., mechanical manipulation without use oforganic solvents) of the hydrophobic component to provide at leastequivalent recovery of nucleic acids in the aqueous solution to producetest sample with less effort and hazard. Hydrophobic components tend tonaturally segregate, e.g., driven by hydrophobic interactions, whenexposed to the aqueous solutions and incubation conditions of thepresent invention. Typically, the hydrophobic component does not havethe same density as the aqueous solution so a hydrophobic layer canform, e.g., above or below the suspension. This can be accelerated oraffected by centrifugation. Such a layer can be separated from theaqueous layer by various physical means, e.g., by physically decantingthe hydrophobic layer off the top, aspirating either the aqueous layeror hydrophobic layer away from the other, pipetting the layers from eachother, solidifying the hydrophobic component at a temperature below themelting point so that it can be physically removed from the aqueouslayer as a solid or semisolid. In preferred embodiments, the separationof the bulk of hydrophobic component from the sample does not includethe use of organic extraction steps before the incubation step and/orafter the incubation step.

Nucleic acids released from cells or tissues by the methods can beexcellent test sample material for input to any number of nucleic acidanalytical techniques. In many cases, the nucleic acids released intothe aqueous solution can be captured on a solid support for detection byvarious assays known in the art. The solubilization methods, typicallyin combination with the complimentary methods further described herein,can be useful to provide accurate quantitation. To further purify thenucleic acids solubilized in the methods, for assays sensitive todisruption by cell lysate constituents, the separated solution can bephenol extracted and ethanol precipitated, as is known in the art. Inpreferred methods, the separated solution is analyzed, e.g., by a bDNAassay, without any organic extraction and/or denaturation steps (butwith physical hydrophobic component separation). The released solutionsof nucleic acids (typically, lysates) can provide good assay inputmaterial for various assays, including, e.g., bDNA analysis, northernblot analysis, Southern blot analysis, polymerase chain reaction;nucleic acid sequencing, agarose gel electrophoresis, differentialdisplay techniques, and the like.

In another aspect of the invention, test cell numbers represented in alysate can be estimated based on the amount of a repetitive DNA that israrely deleted or duplicated in the genome of a cell or tissue. Themethods of determining a number of test cells can include, e.g.,obtaining a reference nucleic acid sample from a known number ofreference cells, quantitating the amount of a ribosomal DNA in thereference sample, providing a standard function (such as, a standardcurve or a standard equation obtained through regression analysis) forthe reference cell number versus the reference ribosomal DNA quantity,obtaining a test nucleic acid sample from test cells, quantitating theamount of the ribosomal DNA in the test sample, and determining the testcell number based on the standard function and the quantity of testribosomal DNA.

Test cells and standard cells for determining cell number by the methodsof the invention can be any of one or more type. The reference cells orthe test cells requiring number determination can be, e.g., tumor cells,cells from a cell line, cells from a microscope slide, FFPE cells,normal cells, polyploid cells, and the like. In certain embodiments, thetest cells have been lung tumor cells or colon tumor cells. In othercases, the test cells providing the reference nucleic acid sample have asubstantially normal karyotype.

The preferred repetitive DNA for normalizations in the methods ofdetermining test cell numbers is a ribosomal DNA. These DNAs are highlyrepetitive and are located on multiple chromosomes at positions lessprone to translocations, deletions or insertions. Therefore, we havefound them to be consistently represented in normal numbers, even ingrossly aneuploid cell lines. In more preferred embodiments, ribosomalDNA is a 18S rDNA, a 5.8S rDNA, and/or a 28S rDNA.

The repetitive DNA can be quantitated (e.g., in a dilution series) fordetermination of a standard function, e.g., by any suitable technique,such as bDNA analysis, Southern blot analysis, polymerase chainreaction, agarose gel electrophoresis, and the like. The results can beused in comparison to test sample results to determine the number ofcells represented in a test sample. For example, determining the testcell number can be by inputting the ribosomal DNA quantity of a testcell sample into the standard function, e.g., inputting the testribosomal DNA quantity into a formula comprising a ratio of cells torDNA, a computer with analytical software; or into a comparison to astandard value on a standard curve. Using the determined test cellnumber, results of other analyses can be normalized to the cellnumber—for example, normalizing an mRNA assay result to copies per cell.

Standard functions determined based on known cell numbers and repetitiveDNA can also be used to determine an efficiency of solubilization whenthe number of cells in a test sample is known. For example, theefficiency of a test nucleic acid extraction can be determined from theknown number of test cells in a solubilized lysate compared to thenumber of test cells calculated from the standard curve to berepresented in the test cell lysate.

In a further aspect of the invention, RNA that has been degraded can bemore accurately quantitated using a degraded in vitro transcribed (IVT)RNA standard curve. For example, mRNA copy numbers can be determined bydetermining a number of cells in a test sample, preparing a standardfunction for an RNA assay output versus a degraded in vitro RNA standardassay input, determining an, amount of a test mRNA in the test sample bythe RNA assay using the standard function, and determining the copynumber of the mRNA in the cells based on the number of cells and thedetermined amount of test mRNA. In a preferred embodiment, theappropriate degraded IVT RNA standard can be selected or the slope of astandard curve can be modified according to the results of an offsetbDNA assay (described below).

In another aspect of the invention, the level of fragmentation of a bDNAtarget nucleic acid can be determined by an assay wherein captureextenders and label extenders are offset from each other along a targetnucleic acid. For example, fragmentation of a target nucleic acid(having a target sequence unknown to be full length or fragmented tosome extent) can have fragmentation detected by analyzing a sample ofthe nucleic acid in an offset bDNA assay wherein each of one or more,offset capture extender probe C3 sequences are complimentary tosequences along the nucleic acid, and where one or more offset labelextender L1 sequences are complimentary to sequences of the nucleic acidat positions spaced at least one nucleotide base either 5′ or 3′ fromall the C3 complimentary sequences. With such a bDNA assayconfiguration, less signal can be generated if the nucleic acid isfragmented between the C3 and L1 complimentary sequences than if thenucleic acid is not fragmented between the C3 and L1 complimentarysequences. For example, less signal can be generated if a significantportion of the nucleic acid is in a fragmented form.

In many cases, the offset assay can be made more accurate and reliableby having an appropriate control assay run on the sample using astandard bDNA assay having interspersed capture extenders and lapelextenders. For example, a sample of the nucleic acid can be analyzed ina second bDNA assay wherein two or more control capture extender probeC3 sequences are complimentary to sequences at different positions alongthe nucleic acid, and where one or more control label extender L1sequences are complimentary to sequences at different positions alongthe nucleic acid sequence with one or more of the control L1 sequencesbeing complimentary to the nucleic acid at positions between thepositions complimentary to two or more of the control C3 sequences. Theratio of the control assay result over the offset assay result will behigher if the nucleic acid is fragmented than the ratio if the nucleicacid is not fragmented.

The offset assay can be more sensitive when the label extenders arefurther offset from the capture extenders along the target nucleic acid.In preferred embodiments, the nucleic acid sequences complimentary, tothe offset C3 sequences are separated from the nucleic acid sequencescomplimentary to the offset L1 sequences by a space of 75% or more ofthe nucleic acid nucleotides (that is, based on the full length targetnucleic acid, the space between the nearest LE and CE sites would be atleast 75% of the target nucleic acid length). Describing the CE/LEspacing another way, it is preferred the offset label extender L1sequences be complimentary to sequences of the nucleic acid spaced atleast 25 nucleotide bases 5′ and/or 3′ from all the C3 complimentarysequences. In preferred embodiments, no L1 complimentary sequence isbetween any two C3 complimentary sequences.

In other aspects of the offset bDNA assay technology, blocking probeshaving sequences complimentary to sequences of the target in the spacebetween LE and CE compliments are included during hybridization, e.g.,to reduce assay background signals.

The output of offset bDNA assays and ratios to controls can characterizethe condition of a sample. For example, the offset assay signal, orratio to control, can be correlated to the average length of the targetnucleic acid sequences. The fragmentation level thus provided can beused, e.g., to select an assay standard or to select a standard functionfor use in RNA quantitation versus a degraded IVT RNA standard.

DEFINITIONS

Unless otherwise defined herein or below in the remainder of thespecification, all technical and scientific terms used herein havemeanings commonly understood by those of ordinary skill in the art towhich the present invention belongs.

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particular processes oranalytical methods, which can, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting. As used in this specification and the appended claims, thesingular forms “a”, “an” and “the” include plural referents unless thecontent clearly dictates otherwise. Thus, for example, reference to “acomponent” can include a combination of two or more components;reference to “a nucleic acid” can include mixtures of nucleic acids, andthe like.

Although many methods and materials similar, modified, or equivalent tothose described herein can be used in the practice of the presentinvention based on the present specification without undueexperimentation, many preferred materials and methods are describedherein. In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set outbelow.

The term “about” as used herein indicates the value of a given quantityvaries by +/−10% of the value, or optionally +/−5% of the value, or insome embodiments, by +/−1% of the value so described.

The term “polynucleotide” (and the equivalent term “nucleic acid”)encompasses any physical string of monomer units that can becorresponded to a string of nucleotides, including a polymer ofnucleotides (e.g., a typical DNA or RNA polymer), peptide nucleic acids(PNAs), modified oligonucleotides (e.g., oligonucleotides comprisingnucleotides that are not typical to biological RNA or DNA, such as2′-O-methylated oligonucleotides), and the like. The nucleotides of thepolynucleotide can be deoxyribonucleotides, ribonucleotides or polymersof nucleotide analogs, can be natural or non-natural, and can beunsubstituted, unmodified, substituted or modified. The nucleotides canbe linked by phosphodiester bonds, or by phosphorothioate linkages,methylphosphonate linkages, boranophosphate linkages, or the like. Thepolynucleotide can additionally comprise non-nucleotide elements such aslabels, quenchers, blocking groups, or the like. The polynucleotide canbe, e.g., single-stranded or double-stranded.

A “polynucleotide sequence” or “nucleotide sequence” is a polymer ofnucleotides (an oligonucleotide, a DNA; an RNA, a nucleic acid, etc.) ora character string representing a nucleotide polymer, depending oncontext. Prom any specified polynucleotide sequence, either the givennucleic acid or the complementary polynucleotide sequence (e.g., thecomplementary nucleic acid) can be determined:

Two polynucleotides “hybridize” when they associate to form a stableduplex, e.g., under relevant assay conditions. Nucleic acids hybridizedue to a variety of well characterized physico-chemical forces, such ashydrogen bonding, solvent exclusion, base stacking and the like. Anextensive guide to the hybridization of nucleic acids is found inTijssen (1993) Laboratory Techniques in Biochemistry and MolecularBiology-Hybridization with Nucleic Acid Probes, part I chapter 2,“Overview of principles of hybridization and the strategy of nucleicacid probe assays” (Elsevier, N.Y.).

The term “complementary” refers to a polynucleotide that forms a stableduplex with its “complement,” e.g., under relevant assay conditions.Typically, two polynucleotide sequences that are complementary to eachother have mismatches (mismatched base pairs) at less than about 20% ofthe bases, at less than about 10% of the bases, preferably at less thanabout 5% of the bases, one mismatch, and more preferably have nomismatches.

A “capture extender” or “CE” is a polynucleotide (or comprises anucleotide) that is capable of hybridizing to a nucleic acid of interestand to a capture probe. The capture extender typically has a firstpolynucleotide sequence C-1, which is complementary to the captureprobe, and a second polynucleotide sequence C-3, which is complementaryto a polynucleotide (target) sequence of the nucleic acid of interest.Sequences C-1 and C-3 are typically riot complementary to each other.The capture extender is preferably single-stranded.

A “capture probe” or “CP” is a polynucleotide that is capable ofhybridizing to at least one capture extender and that is tightly bound(e.g., covalently or noncovalently, directly or through a linker, e.g.,streptavidin-biotin or the like) to a solid support, a spatiallyaddressable solid support, a slide, a particle, a microsphere, a bead,or the like. The capture probe typically comprises at least onepolynucleotide sequence C-2 that is complementary to polynucleotidesequence C-1 of at least one capture extender. The capture probe ispreferably single-stranded.

A “label extender” or “LE” is a polynucleotide that is capable ofhybridizing to a nucleic acid of interest and to a label probe system.The label extender typically has a first polynucleotide sequence L-1,which is complementary to a polynucleotide sequence of the nucleic acidof interest, and a second polynucleotide sequence L-2, which iscomplementary to a polynucleotide sequence of the label probe system(e.g., L-2 can be complementary to a polynucleotide sequence of anamplification multimer, a preamplifier, a label probe, or the like). Thelabel extender is preferably single-stranded.

A “label” is a moiety that facilitates detection of a molecule (e.g., byproviding a detectable signal). Common labels in the context of thepresent invention include fluorescent, luminescent, light-scattering,and/or colorimetric labels. Suitable labels include enzymes andfluorescent moieties, as well as radionuclides, substrates, cofactors,inhibitors, chemiluminescent moieties; magnetic particles, and the like.Patents teaching the use of such labels include U.S. Pat. Nos.3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and4,366,241. Many labels are commercially available and can be used in thecontext of the invention.

A “label probe” or “LP” is a single-stranded polynucleotide thatcomprises a label (or, optionally, that is configured to bind to alabel) that directly or indirectly provides a detectable signal. Thelabel probe typically comprises a polynucleotide sequence that iscomplementary to the repeating polynucleotide sequence M-2 of anamplification multimer; however, if no amplification multimer is used inthe bDNA assay, the label probe can, e.g., hybridize directly to a labelextender.

A “label probe system” comprises one or more polynucleotides thatcomprise one or more labels and one or more polynucleotide sequencesM-1, each of which is capable of hybridizing to a label extender. Thelabel provides a signal, directly or indirectly. Polynucleotide sequenceM-1 is typically complementary to sequence L-2 in the label extenders.The one or more polynucleotide sequences M-1 are optionally identicalsequences or different sequences. The label probe system can include aplurality of label probes (e.g., a plurality of identical label probes)and an amplification multimer; it optionally also includes apreamplifier or the like, or optionally includes only label probes, forexample.

An “aqueous solution”; as used herein, refers to an aqueous solution(water containing one or more solutes) suitable to retain a nucleic acidof interest in solution. For example, an aqueous solution can have a pHand ionic strength conducive to dissolving and holding a nucleic acid insolution. Aqueous solutions of the invention can optionally includeconstituents useful in releasing nucleic acids from cells and tissues,such as, e.g., pH buffers, salts, surface active agents and/orproteases. Aqueous solutions optionally include constituents that areuseful as components of a nucleic acid assay or hybridization, such as,e.g., formamide, a sodium chloride-sodium citrate (SSC) buffer, nucleicacid probes, and the like.

A “hydrophobic component” associated with a cell or tissue sample is acompound substantially insoluble in water (e.g., less than 1% soluble inpure water). Typical hydrophobic components associated with samples arelipids, fats, oils, hydrocarbons, waxes, hydrophobic membranecomponents, and the like. In certain embodiments of the invention, thehydrophobic component is a paraffin, e.g., clinical sample embeddingwax.

“Physically separating”, as used herein, refers to separation of ahydrophobic component layer from an aqueous solution layer by physicalmeans. Organic extraction of a hydrophobic component from an aqueoussuspension of a sample is considered a chemical separation of thehydrophobic component, e.g., from a FFPE sample and is not considered tobe physical separation, even at the point when the organic extractionphase is removed from the remaining aqueous phase. Physical separationis generally a mechanical procedure to remove the hydrophobia component(in solid, semisolid or liquid form) from contact with the aqueoussolution, e.g., by grasping, pushing, sucking, aspirating, pouring,blowing, filtering, adsorbing, absorbing, pulling, and/or the like.Separation can include removing the hydrophobic component from theaqueous solution or removing the aqueous solution from the hydrophobiccomponent.

As used herein, a “standard function” is a function, or expression of afunction, that represents a relationship between two assay parameters,such as, e.g., a known assay input and the resulting output. The outputcan be a raw data output or a value (such as, a number of molecules orconcentration of an analyte) derived from the output. Standard functionsand their, expressions (e.g., standard curves) are well known in theart. The standard function can be in the form of an algebraic function(e.g., equation for a line) or can be provided in the form of a standardcurve (e.g., resulting from regression analysis) on an X-Y chart. Thestandard function can also be expressed as a ratio, constant, oralgorithm (e.g., in the form of computer software).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a block flow diagram of a prior art dewaxing protocol usingorganic solvents (e.g., Dewax) and phase extraction techniques to removeparaffin from formalin fixed paraffin embedded clinical samples beforeinitiation of nucleic acid solubilization “homogenization” protocols.

FIG. 2 shows an exemplary solubilization (“homogenization”) protocol ofthe invention wherein preliminary dewaxing of the sample is not requiredand paraffin is ultimately separated from the aqueous nucleic acidhomogenate (lysate) by a single non-extraction, non-denaturing physicalseparation step.

FIG. 3 shows a graphic representation of mRNA expression induction.Fold-induction determinations of LDHA mRNA levels in tumor cells overnormal cells are found to be substantially the same usingpost-Solubilization separation of hydrophobic components from lysate asfor the old art organic phase extraction before homogenization (lysatepreparation).

FIGS. 4A to 4E show examples of ribosomal DNAs (rDNAs) retainingsubstantially consistent representation in various normal cells,aneuploid cells, and tumor cells.

FIG. 5 shows bDNA assay standard curves demonstrating a linear responsebetween cell line cell number and assay signal output for rDNAs.

FIG. 6 shows a schematic flow diagram of a protocol to establish a cellnumber standard curve based on rDNAs.

FIG. 7 shows standard curves of cell numbers versus bDNA assay outputfor 185 and 28S ribosomal DNAs.

FIG. 8 shows a standard curve of cell number versus 18S DNA. Histogramsshow the 18S DNA standard curves can provide substantially accurate cellnumber determinations, even for aneuploid cell lines.

FIG. 9 shows quantitation of cell numbers obtained by bDNA assay of 18SDNA for 49 tumor tissue scrapings from FFPE slides.

FIG. 10 shows gel electrophoresis of intact full length in vitrotranscribed (IVT) RNAs and hydroxide degraded IVT RNAs.

FIG. 11 shows standard curves for RNA attamole input to a bDNA assayverses assay signal output for full length or degraded IVT transcribedRNA standards. A standard equation is presented for the relationshipbetween assay output for intact versus degraded RNA.

FIG. 12 shows intact and degraded IVT RNA standard curves for 6 mRNAs.

FIG. 13 shows raw assay output data reflecting expression of 6 mRNAs in4 formalin fixed, paraffin embedded, cell lines.

FIG. 14 shows copy numbers per cell for 6 mRNAs calculated based onintact or degraded IVT RNA standard curves.

FIG. 15 shows raw bDNA assay output data for 6 mRNAs determined in 49human tumors.

FIG. 16 shows a chart displaying the determined mRNA copy number percell, based on degraded IVT. RNA standards, for 6 mRNAs in 49 tumor cellsamples.

FIG. 17 shows a schematic diagram of an exemplary bDNA assay system.

FIGS. 18A and 18B show schematic diagrams of offset probe and controlschemes for detecting target nucleic acid fragmentation. Target RNA inFIG. 18A is unfragmented and thus a signal can be generated from labelextenders retained at the solid support using either the control probesystem or the offset probe system. Target RNA in FIG. 18B is fragmentedbetween CE anchoring points and LE complimentary sites for the offsetprobe system, so a signal would not be generated by the bDNA assay.Meanwhile, for the same fragmented target RNA sequence in the controlsystem, CE anchoring points and LE complimentary sites exist on bothsides of the break, so a signal can still be generated for a bDNA assayusing the control probe system.

FIG. 19 shows how the ratio of control to offset probe signals can becorrelated to RNA degradation, as visualized on an agarose gel.

DETAILED DESCRIPTION

The present invention is directed to methods of collecting andquantitating nucleic acids from cells and tissues. Cells, previouslyprocessed, e.g., by formalin fixation and paraffin embedding, can besuspended in an aqueous homogenization solution at elevated temperaturesunder non-denaturing conditions so that nucleic acids are released fromcells into a lysate solution separate from the paraffin material. Thenumber of source cells and the efficiency of nucleic acid extraction canbe accurately determined by a normalization based on a ribosomal DNAreference. The copy number of an mRNA per cell can be more accuratelydetermined using analyses normalized with an appropriately degraded invitro transcribed (IVT) RNA standard material. An estimation of sampleRNA degradation can be provided, e.g., by comparing bDNA signals fromcontrol assays wherein capture and label sites are dispersed along anRNA target against a signal for a test assay wherein capture sites areon one end of the target RNA separated from label sites on the other endof the target RNA, as will be discussed below.

Prior art methods of determining nucleic acids in embedded clinicalsamples required repeated organic chemical extractions (dewaxing) beforesolubilization of nucleic acids from the sample. The present inventioneliminates the requirement for such an extraction step altogether.Scraped-off sections or chunks of tissue in a paraffin block can bedirectly solubilized by, e.g., adding 3004 Homogenizing Solution(Panomics Fremont Calif.) supplemented with Proteinase K (0.3 mg/mL) per60-100 microns (25-250 mm²) pooled tissue sections or 10 mg unsectionedtissue from a block and digested overnight at 65° C. Paraffin than, canseparate from the tissue homogenate during overnight Proteinase Kdigestion at 65° C. and, if abundant, forms a visible layer aboveaqueous homogenate. Liquid paraffin can be physically removed with apipettor or allowed to solidify at room temperature duringcentrifugation, pierced, and the solubilized material (lysate)physically removed from underneath the hydrophobic component, e.g., byaspiration. The aqueous lysate solution can be transferred to a freshmicrofuge tube for immediate use or stored at −80° C. for future use.The present invention significantly improves the accuracy and simplifiesquantification of RNA or DNA from formalin fixed paraffin embedded(FFPE) samples.

A major difficulty in studying differential gene expression by cells,such as those of FFPE samples, is how to normalize for geneheterogeneity. The gene transcript number (mRNA copies) is typicallyexpressed as a copy number per cell. Unfortunately, counting of cells isnot practical for many FFPE tissues or cell slides. In many cases, thecopy number calculations have been normalized based on total DNA ortotal RNA recovered from the sample. However, total cellular DNA and RNAcontent can increase or decrease, e.g., with tumor aneuploidy (Jacques Bde Kok, Rian W Roelofs, Belinda A Giesendorf, Jeroen L Pennings, Erwin TWaas, Ton Feuth, Dorine W Swinkels and Paul N Span, LaboratoryInvestigation (2005) 85, 154-159). Thus, it has not been known how toaccurately quantify the number of cells represented in a FFPE sample. Wehave found that one possibility is to normalize based on a gene or genesidentified as not affected by tumor aneuploidy. Preferably, the genesare represented as multiple copies in the genome to increase thesensitivity of detection. Furthermore, it is preferred that the gene orgenes be stable and distributed across multiple chromosomes, so thatloss or gain of some of the genes on any one chromosome would have lessof an effect on the overall cell number determination.

About 30% of the human genome consists of repeated sequences (Britten,R. J. & Kohne, D. E. (1968) Science 161, 529-540), of which over halfare repeated more than 10⁵ times per genome. Some of these repeatedsequences are organized in long, tandem arrays, while others areinterspersed among less frequently represented sequences includingstructural genes. A number of human interspersed repeated DNA sequencefamilies have been identified. The largest family consists of 3×10⁵copies of related sequences, each about 300 nucleotides long, with mostmembers having a site that can be cleaved by the restrictionendonuclease Alu I (Rubin, C. M., Houck, C. M., Deininger, P. L.,Friedmann, T. & Schmid, C. W. 1980 Nature 284, 372-374). Other, lessfrequently represented, short, interspersed repeated sequences (SINES)have been reported by Deininger et al. (Deininger, P. L., Jolly, D. J.,Rubin, C. M., Friedmann, T. & Schmid, C. W. (1981) J. Mol. Biol. 151,17-33.) and Miesfeld et al. (Miesfeld, Krystal, M. & Arnheim, N. (1981)Nucleic Acids Res. 9, 5931-5947). Adams et at (Adams, J. W., Kaufman, R.E., Kretschmer, P. J., Harrison, M.& Nienhuis, A. W. (1980) Nucleic.Acids Res. 8, 6113-6127) reported a longer interspersed repeated DNAsequence (LINES) family that is 6,400 nucleotides long on the averageand are represented some 4×10³ times per genome. These repetitive DNAsequences are known to mediate or enhance the rate of recombination inthe genomes of many organisms (Jelinek and Schmid 1982; Hardman 1986;Vogt 1990), whereby the interspersed repetitive DNA consists of repeatunits dispersed throughout the genome. Mispairing between such repeatshas been shown to be a frequent cause of deletions and duplications(Smita M. Purandare, and Pragna I. Patel, 1997 7: 773-786 Genome Res.)and thus are often not practical to use to determine the cell number inFFPE tissue samples.

Of more practical use are the human 5.8S, 185 and 28S ribosomal RNA(rRNA) genes (rDNA), present at ˜800 copies per human diploid genome,clustered on the P12 short arms of the five acrocentric chromosomes 13,14, 15, 21, 22; for 10 clusters per diploid genome of 46 chromosomes anda total of 6.4×10⁹ bp. (Worton, R. G., Sutherland, J., Sylvester, J. E.,Willard, F. H., Bodrug, S., Dube, I., Duff, C., Kean, V., Ray, N. P. andSchmickel, R. D. (1988), Science, 239, 64-68). Each ribosomal gene ispart of a 43 kb repeat unit that can be divided into two regions: a 13.3kb transcribed region which contains the highly conserved genes for 18S,5.85 and 28S rRNA subunits of the ribosome, and a 30 kb non-transcribedspacer (NTS) (Gonzalez, L. I., Wu, S., Li, W., Kuo, A. B. and Sylvester,E. J. (1992) Nucleic Acids Res., 20, 5846-5847.) Repeat unit clustersconsist of head-to-tail arrays of ˜80 repeats (Sakai, K., Ohta, T.,Minoshima, S., Kudoh, J., Wang, Y., De Jong, J. P. and Shimizu, N.(1995)).

The NCBI has established the SKY/M-FISH and CGH database to provide apublic platform for investigators to share and compare their molecularcytogenetic data. The karyotypes of hundreds of tumors can be viewed onthe SKY/comparative genomic hybridization database website. Thisdatabase is a part of the Cancer. Chromosome Aberration Projectsponsored by the National Cancer Institute (Kirsch, I. R, Green, E. D.,Yonescu, R., Strausberg, R., Carter, N., Bentley, D., Leversha, M. A.,Dunham, I., Braden, V. V., Hilgenfeld, E., Schuler, G., Lash, A. E.,Shen, G. L., Martelli, M., Kuehl, W. M., Klausner, R. D., and Ried, T.Nat. Genet., 24:339-340, 2000). In addition, the complete karyotypes for59 cell lines can be viewed on the interne, including representativeimages of the cell line karyotypes (Anna V. Roschke, Giovanni Tonon,Kristen S. Gehlhaus, Nicolas McTyre, Kimberly J. Bussey, Samir Lababidi,Dominic A. Scudiero, John N. Weinstein, 2 and Ilan R. Kirsch CANCERRESEARCH 63, 8634-8647, 2003).

After viewing individual tumor karyotypes (lung, colon, breast, etc.)and 59 cell line karyotypes for their ribosomal DNA content, wedetermined here that ribosomal DNA gene content varied very little inthe primary tumors, which is do in part to the fact that the ribosomalgenes are clustered on the P12 short arms of the five acrocentricchromosomes and thus the loss or gain of any one short arm equals only10% divergence from the normal tissue ribosomal gene dosage. Since theploidy and total DNA of each cell line and tumor varies, while theribosomal DNA number varies less, we have found that more accurate cellnumber estimates can be obtained using normalization by ribosomal DNA.

In another aspect of the invention, an mRNA copy number for cells, e.g.,previously embedded in a microscope slide, can be determined withenhanced accuracy by normalization according to a similarly degraded RNAstandard. The cell number represented in the post-homogenizationseparated solution of nucleic acids from the embedded cells can bedetermined based on a standard function (e.g., a standard curve) of cellnumber versus a quantity of repetitive DNA (such as a ribosomal DNA).The amount of the mRNA can be determined for the solution of nucleicacids based on, e.g., a standard curve of degraded IVT RNA versus an RNAassay (such as, e.g., bDNA) output. The accuracy of such a copy, numberdetermination can be improved by consideration of extraction efficiencyfrom the repetitive DNA data and mRNA normalization by the degraded RNAcurve, as discussed herein.

For many, methods of the invention, bDNA technology is a preferrednucleic acid quantitative technique: bDNA technology is fortunate in notbeing subject to many of the problems of QPCR in quantitation of RNAs.In fact the hybridization step in bDNA assays can be facilitated byformalin-modification of RNA. In addition, impurities that reduce theactivity of reverse transcriptase and DNA polymerase in QPCR can beinconsequential in the branched chain assay since this assay does notrequire enzymatic activities, but relies instead on hybridilation. Toovercome the problems of RNA degradation and fragmentation, a uniqueapproach of probe design has been developed for the bDNA assay (WenYang, Botoul Maqsodi, Yunqing. Ma, Son Bui, Kimberly L. Crawford, GaryK. McMaster, Frank Witney, and Yuling. Luo, Direct quantification ofgene expression in homogenates of formalin-fixed; paraffin-embeddedtissues Biotechniques, Vol. 40, No. 4 (2006), pp 481-486; Warrior U, FanY, David C A, Wilkins J A, McKeegan E M, Kofron J L, Burns D J:Application of QuantiGene nucleic acid quantification technology forhigh throughput screening, J Biomol Screen 2000, 5:343-352; Bushnell S,Budde. J, Catino T, Cole J, Derti A, Kelso R, Collins M L, Molino G;Sheridan p, Monahan J, Urdea M: ProbeDesigner: for the design ofprobesets for branched DNA (bDNA) signal amplification assays,Bioinformatics 1999, 15:348-355. Many methods of the present inventionprovide ways to improve bDNA analyses; and other quantitative nucleicacid assays, with regard to problematic samples.

Collecting Nucleic Acids from FFPE Tissue Samples

In order to obtain sensitive, quantitative and reliable assay resultsfor nucleic acids from cells and tissues, it is important to harvest auseful amount of the nucleic acid substantially free of physically andchemically interfering substances. This problem can be particularlydifficult for samples, such as paraffin embedded clinical samples thatare associated with hydrophobic preservatives and waxy supportingmatrices. In the present invention, we have found that reliable andquantitative separation of cells and tissue contents from hydrophobicsample components can be accomplished, e.g., by breaking the sample intoparticles of small size, suspending the particles in an appropriatelysis solution containing a protease, incubating the suspension at atemperature above the melting temperature of the hydrophobic componentto release the nucleic acids into an aqueous phase and generate aseparate hydrophobic component phase, and separating the aqueous phasefrom the hydrophobic phase.

Obtaining Small Particles of the Cell or Tissue Sample

If the cell or tissue sample is not already in the form of smallparticles or sheets with a high surface to volume ratio, it is usuallybeneficial to break the sample into smaller bits so that nucleic acidscan be extracted into an aqueous solution in a relatively quantitativeand timely fashion. Samples, such as dried cell bulks, embedded tissues,formalin fixed paraffin embedded cells or tissues, clinical samplesstored on microscope slides, and the like, can be reduced to a finepowder or paste, using any appropriate methods known in the art, toenhance the harvest of nucleic acids from the sample.

For example, the samples can be chopped, ground, milled, scrapped,dounced, homogenized, sheared, and/or the like, to particle sizesreasonably adapted to nucleic acid release from cells and tissues by themethods of the invention. For example, the samples can be broken intoparticles of average diameter less than 1 mm, less than 0.1 mm, lessthan 10 um, less than 1 um, or less. In preferred embodiments, thesamples are broken down to particles about 100 times the volume of thecells; 10 times the volume of the cells, or about the size of the cells.

The samples can be physically and/or chemically broken down to theappropriate size in the presence of a liquid matrix, or not. The liquidmatrix can be an aqueous solution with components suitable forintroduction into later handling and/or analysis steps. For example theliquid matrix can provide a suitable environment for an enzymaticreaction or a stringent nucleic acid hybridization.

Suspension of Particles in an Aqueous Solution

Sample particles are placed in an aqueous solution for treatments thatrelease nucleic acids from the sample cells into the solution forming alysate. The aqueous solution can be as simple as water to dissolve thenucleic acids. Typically the aqueous solution includes ingredients thatincrease the solubility of the nucleic acids, disintegrate cell/tissuestructures that interfere with release of the nucleic acids, and/orprovide a suitable environment for analysis of the nucleic acids.

The aqueous solution can include constituents useful to later analyticalprocedures and/or storage conditions. For example, the aqueous solutioncan include constituents of a nucleic acid hybridization buffer, e.g.,PEG, SDS, SSC buffer, NaHPO4, EDTA, denatured salmon sperm DNA,formamide, SSPE, etc. The aqueous solution can include bDNAconstituents, such as, e.g., blocking probes, capture extenders, labelextenders, preamplifiers, label probes, amplification probes,amplification multimers, and the like. In preferred embodiments forrelease of nucleic acids from many formalin treated or paraffin embeddedsamples, the aqueous solution can include one or more of: a protease,lipase, surfactant, or nuclease inhibitor.

The sample particles can be suspended in the aqueous homogenizationsolution while the sample is being broken up into small particles. Forexample, the sample can be ground, milled or dounced in the presence ofthe desired aqueous solution. Alternately, the sample can be, e.g.; dryground and transferred into a aqueous solution after particle sizing.Alternately, the particles can be, e.g., centrifuged or filtered from aliquid matrix used for sizing the particles and exchanged over to thedesired aqueous solution for suspension and release of nucleic acids(e.g., by dialysis, diafiltration, resuspension after centrifugation orfiltration, etc.).

The sample particles can be suspended in the aqueous solution to exposethe particles to conditions that release nucleic acids. The suspensioncan be made by, e.g., stirring, douncing, vortexing, inverting, mixing,shaking, or simply by introducing the particles to the solution.Although the mixture often starts as a suspension of particles, most orall of the sample material typically ends up in solution or in ahydrophobic layer by the end of suspension and incubation treatments.

In preferred embodiments, the aqueous solution is a solvent for nucleicacids and is an appropriate assay solution for an intended nucleic acidassay. For example, the aqueous solution can provide conditions of pH,ionic strength, viscosity, surface active agents, blocking agents, etc,suitable for attachment of the nucleic acids to a solid support (such asa blotting membrane) or for stringent hybridization of the nucleic acidwith one or more nucleic acid targets or probes.

In many cases, it is desirable to include one or more proteases in theaqueous homogenization solution. Significant amounts of the desirednucleic acid are often entrapped by the proteins and protein matrices ofcells and tissues. Proteases can help disrupt these proteins and helprelease the nucleic acids. In preferred embodiments, the protease is aproteinase K, e.g., at a concentration of more than 50 ug/ml, 100 ug/ml,150 ug/ml, 200 ug/ml, 300 ug/ml, 500 ug/ml, or more.

Incubating the Suspension

The suspension of the sample in the aqueous solution can be incubated ator above the melting temperature of one or more sample hydrophobiccomponents. The incubation can melt and release the hydrophobiccomponent from the sample material and disintegrate cell and/or tissuestructures to form an aqueous lysate containing nucleic acids ofinterest.

Hydrophobic components of samples can include lipids, fats and/or oilsnaturally present in the sample of cells or tissue, or not. Hydrophobiccomponents typically of most concern in analysis of nucleic acids fromclinical samples are preservative and embedding compositions processedinto the sample to aid in the preservation, handling, and/or storage ofthe samples. Commonly encountered hydrophobic components with regard tosamples used in methods of the invention are cell and tissue embeddingmedia, such as paraffin embedding waxes.

Incubation of the suspension is preferably under conditionsnon-denaturing to the genomic DNA of the sample. The conditions oftemperature, ionic strength, pH, divalent cation concentration,formamide concentration, and the like, well known in the art, can besignificantly less than conditions that would denature (melt) apreponderance of the DNA in the sample (see Rapley, R. and Walker, J. M.eds., Molecular Biomethods Handbook (Humana Press, Inc. 1998). Forexample, the Tm of a DNA-DNA duplex can be estimated using the followingequation:

Tm(° C.)=81.5° C.+16.6(log₁₀ M)+0.41(% G+C)−0.72(% f)−500/n,

where M is the molarity of the monovalent cations (usually Na+), (% G+C)is the percentage of guanosine (G) and cystosine (C) nucleotides, (% f)is the percentage of formamide and n is the number of nucleotide bases(i.e., length) of the hybrid. Tm increases with higher ionicconcentrations of the solvent due to the stabilizing effects thatcations have on DNA duplex formation. More cations bind to duplex DNAthan to the component single strands. Different cations may havedifferent effects on Tm. The most common monovalent cation is Na+;however, from a Tm standpoint, sodium and potassium are functionallyinterchangeable. Divalent cations (such as Mg++) also stabilize DNAhybrids (increase Tm) but their effects are quantitatively muchdifferent from monovalent cations. Increased pH can increase chargerepulsion between DNA strands and thus lower the Tm. In preferredembodiments, the incubation temperature during solubilization is at leak2° C., 5° C., or 10° C. below the Tm for DNA of the sample in thesuspension.

The suspension can be incubated at a temperature above the meltingtemperature of one or more hydrophobic components present in the sample.For example, the suspension can be incubated at a temperature rangingfrom about 40° C. to about 100° C., from about 41° C. to about 95° C.,from about 45° C. to about 90° C., from about 50° C. to about 80° C.,from about 60° C. to about 70° C., or about 65° C. Preferred incubationtemperatures are at least above the melting temperature of the highestmelting hydrophobic component or the most abundant hydrophobic componentin the sample. Preferred incubation temperatures are temperatures alsosupporting the desired activity of enzymes, such as proteases in thesolution. For clinical samples embedded in paraffin, the melting pointcan be, e.g., between about 43° C. and 71° C., more commonly between 52°C. and 64° C., depending on chain lengths and degree of refinement. Wehave found that most embedded clinical samples include paraffin that isreadily melted and separated from the aqueous solutions at a temperatureof 65° C.

Suspensions of samples can be incubated for a time adequate to releasesufficient amounts of a nucleic acid to carry out a desired analysis.The time can vary depending on, e.g., the amount of connective fibers ina tissue, the incubation temperature, the activity of proteases, theamount of paraffin present, the amount and type of surface active agentspresent, the presence of physical factors (such as agitation), thesensitivity of the desired nucleic acids (and the presence or absence ofdestructive nuclease enzymes and/or their inhibitors), pH, ionicstrength, and the like. Suspension incubation times can range, e.g.,from less than about 30 minutes to 5 days, 3 hours to 3 days, from about6 hours to about 2 days, from about 9 hours to about 1 day, or about 12hours (e.g., over night). We have found that high yields ofapproximately 90% nucleic acid release can be accomplished in over nightincubations, according to methods of the invention.

Physically Separating the Aqueous Solution from the HydrophobicComponent

In methods of the invention, the hydrophobic component is repelled bythe aqueous solution, and thus tends to self-segregate under theinfluence of the solutions and incubation conditions of the invention.Moreover, the hydrophobic component typically has a density different(e.g., lower) from the aqueous solution, and thus will tend to floatabove the aqueous solution, e.g., when released from the sample bymethods of the invention. Once the hydrophobic component is segregatedfrom the rest of the suspension, the hydrophobic component can bephysically separated from sample constituents remaining in the aqueoussolution.

In some cases, the hydrophobic component can be separated from theaqueous lysate solution containing released nucleic acids afterincubation, by simply aspirating it off the top of the incubationcontainer. Alternately, the incubated suspension can be chilled to atemperature below the melting point of the hydrophobic component and itcan be mechanically removed as a solid or the lysate efficientlydecanted or aspirated from under it.

It is often useful to centrifuge the incubated suspension into separatelayers with different densities. For example, an incubated suspensioncan be centrifuged at from 1000×g to about 20,000×g for from 1 minute toabout 1 hour to separate the suspension into, e.g., a bottom cell/tissuedebris layer, a middle lysate layer and a top hydrophobic componentlayer. Such centrifugation can have the benefit of more discretely andmore quantitatively segregating hydrophobic components that may be inthe form of a colloid or suspension of fine lipid globules.Centrifugation can provide discrete layers, such as clarified lysatelayers, that can be readily removed by routine liquid handlingprocedures, e.g., without resort to chemical extraction procedures.

The lysate of aqueous solution with solubilized cell/tissueconstituents, including nucleic acids, can be separated from thehydrophobic component, and from any cell debris, by other techniquesknown in the art. For example the incubated suspension can be filteredthrough a membrane of appropriate materials and pore size. Debris and/orsolidified hydrophobic component can be retained by the filter membranewhile clarified lysate passes through the membrane. Wet hydrophilicmembranes can pass the lysate while retaining the hydrophobic componentdue to hydrophobic repulsion. Hydrophobic membranes can pass the lysatewhile adsorbing and retaining the hydrophobic component.

In some embodiments, hydrophobic components segregated from the sampleduring incubation of the suspension can be removed in an organicextraction (chemical separation), such as a xylene or phenol extraction.In preferred embodiments, separating the hydrophobic components does notrequire an organic extraction step. This is often the case when, e.g.,the lysate is to be used in a bDNA assay.

Lysates obtained from samples according to the methods of the inventioncan be compatible test materials for analysis by any number of nucleicacid assay techniques. In many cases, intelligent selection of aqueoussolution constituents and separation techniques can result in samplesready for direct input to assays, such as bDNA analysis, northern blotanalysis, Southern blot analysis, polymerase chain reaction,spectrophotometry, fluorometry, nucleic acid sequencing, agarose gelelectrophoresis, and the like. In other cases, the lysate can beadjusted as necessary, e.g., buffer exchange procedures or by additionof buffers, substrates, etc., to accommodate a particular assay. In thecase of FFPE lysates, accuracy of assay results can be enhanced, e.g.,by normalization for sample extraction efficiency and/or analytedegradation using techniques of the invention described below.

Quantitation of Nucleic Acids Based on a Multicopy DNA Standard

When cell lysates are prepared from clinical samples, particularlytissue samples or embedded samples, it may be unclear how many cellshave released their contents into the lysate. Although it has been knownto normalize a lysate harvest according to the amount of total DNApresent, this determination can introduce errors, e.g., due toinconsistent DNA content among various cells. We have found that certainrepetitive genes, spread among various chromosomes, and preferably onthe short arms of acrocentric chromosomes can be used to provideconsistent and accurate cell number estimates, even in the context ofcertain aneuploid cells, such as cell lines and tumor cells.

Cell numbers represented in a lysate can generally be better estimatedby normalizing relative to a repetitive gene (preferably a ribosomalgene) that exists on two or more different chromosomes at a site nearthe centromere (such as on a short arm of an acrocentric chromosome). Itcan be beneficial if the repetitive gene exists in a large number ofcopies, e.g., to enhance sensitivity of their detection. Here, we haveidentified ribosomal genes, particularly genes encoding 18S, 285 and5.8S ribosomal RNAs, as excellent reference genes for normalizing cellnumbers represented in lysates, e.g., of common aneuploid cells.

The methods of using rDNAs to provide a cell count can be employed inassociation with any number of quantitative assays involving any cellconstituent. For example, the molecular copy number of any analyte(e.g., a nucleic acid, a protein, or a small molecule) can be calculatedbased on the assayed analyte quantity and cell number derived based onrDNA analysis.

Providing a Standard Function of Cell Number to rDNA

To determine the number of test cells represented in a lysate, one can,e.g., obtain data from which to derive a standard function of ribosomalDNA (rDNA) versus numbers of cells, and interpolate the number of cellsrepresented in an unknown lysate based on the amount of the rDNApresent. A standard function can be an equation expressing therelationship between one quantity and another, such as, e.g., an assayinput and assay output, or a constant proportional relationship betweena number of cells and an amount of RNA in a lysate of the cells. Typicalstandard functions can include, e.g., a standard curve plotting X-Ycoordinates of related values on a chart, an equation established byregression analysis of standard assay results, or a constant ratio orproportion between related parameters. An expression of a standardfunction can be a “best fit” line on a paper chart; a ratio or lineslope representing a proportionality between the cell numbers and theirrDNA; an equation determined by regression analysis techniques; a resultprovided by a computer using an appropriate program, and the like, as isknown in the art.

For example, the number of cells represented in a test lysate can bedetermined by: obtaining a reference lysate from a known number ofcells; quantitating the amount of genes encoding a ribosomal RNA in thereference lysate; determining a ratio of cell numbers to an amount ofthe rDNA in a sample; quantitating the amount of the rDNA is a lysate oftest cells; and, calculating the number of test cells represented in thetest lysate based on the ratio.

The number of cells in a reference sample can be determined, e.g., bycounting them using methods known in the art. For example referencecells grown in suspension can be counted in a hemocytometer, in aCoulter counter, by a cell sorter, inferred by packed cell volume, andthe like. Cells in a reference tissue can be counted microscopically,inferred from tissue volume, or counted as for suspended cells aboveafter release by mechanical, chemical and/or enzymatic techniques. Thereference cells can be normal cells, primary culture cells, cell lines,cells released from tissues, cells from biological fluids, and/or thelike. The reference cells can be the same type as the test cells, ornot. The cells can be uniformly the same or, a mixture of different celltypes.

The test cells enumerated by the standard curve can be any type ofinterest. The test cells can be the same type of cells as the referencecells or at least derived from the same species of animal as thereference cells. In a preferred embodiment, a particular benefit isobtained wherein the test cells do not have to be the same type of cellsas the reference cells. For example, in certain embodiments, thereference cells can be normal cells or mixtures of a variety of cells,while the test cells are aneuploid cells, cells from “immortal” celllines, cancer cells, tumor cells, and the like.

Quantitating Nucleic Acids and Test Cell Numbers

Quantitating rDNAs in lysates to determine cell counts can be by anymethod with sufficient sensitivity and accuracy to provide a usefuloutput. However, because errors in determination of both the referencerDNA and test rDNA carry over to contribute to the error of the finalcell count result, it is preferred the nucleic acid quantitation methodbe precise and accurate. Typically, the rDNA determinations for bothtest and reference samples should use the same methodology, to avoidinterassay variables, but the methods do not have to be the same.

Quantitation of rDNA in the present methods can be by, e.g., QPCR, bDNAanalysis, Northern blot analysis, in situ hybridizations, and the like.With regard to determinations for lysates from formalin fixed, paraffinembedded samples, it is preferred to use bDNA techniques, which are notas sensitive to sample degradation and impurities.

Extraction Efficiency Calculations

In addition to determining unknown test cell counts described above (bycomparing test lysate rDNA to the cell number versus rDNA according to astandard function), an extraction efficiency can be determined for lysisof test cells, based on the same standard curve.

In a situation where the number of test cells is known (e.g., bycounting methods described above), the extraction efficiency can bedetermined for lysis of those cells. For example, the amount of an rDNAcan be determined for the test lysate and the number of cellsrepresented read from a standard curve, prepared as described above. Thepercent extraction efficiency can be calculated as 100 times the numberof cells represented in the test lysate divided by the known number ofcells processed to make the lysate. Such information can be useful,e.g., in optimizing a lysis technique or to normalize an analyticalresult.

Suppose a lysate of cells is to be assayed for the presence of ananalyte and it is important how much of the analyte is present per cell,then the extraction efficiency can be used to improve the accuracy ofthe calculation by normalizing the analyte quantity to the actual numberof cells. For example, in many circumstances, it is useful to know theexpression levels of certain genes in cells. A known number of the cellscan be lysed and a mRNA transcribed from the gene can be quantitated,e.g., by RT-PCR or bDNA methods. rDNA values for the lysate can becompared to a standard curve of cell number versus rDNA to find thenumber of cells represented in the lysate. The extraction efficiency canbe expressed as the ratio of represented cells over the known number ofcells. The amount of the mRNA per cell can be calculated as the totalmRNA in the lysate divided by the known number of cells times theextraction efficiency.

Using a Degraded IVT RNA Curve to Determine mRNA Copy Numbers

Assay of degraded samples for nucleic acids often provides erroneousresults. In particular, analysis of nucleic acids from aged samples orsamples exposed to harsh treatments often results in weak signals andincorrectly low output values or false negative assay output. To solvethis problem, we have determined that standard curves prepared usingdegraded in vitro transcribed (IVT) RNA can improve the accuracy of RNAassays carried out on such degraded samples.

Standard Functions for Degraded RNA

A standard function can be established to represent the relationshipbetween the input of known amounts of a degraded IVT RNA and the outputof a quantitative RNA assay. An unknown RNA, typically known or expectedto be in a degraded form, can then be analyzed using the RNA assay toprovide an output value. When the assay output for the unknown RNA isinput to the standard function, the result can be a more accurate RNAquantity value than for a standard function provided, e.g., byregression analysis of a standard curve prepared using full lengthundegraded IVT RNA standard. If the unknown RNA sample came from a knownnumber of cells, dividing the RNA quantity by the number of cells canprovide a more accurate determination of the RNA copy number per cell.

A standard function can express the relationship between an amount ofdegraded RNA and the output of an RNA assay. The RNA assay can be anyknown in the art, such as, e.g., bDNA analysis, northern blot analysis,RT-polymerase chain reaction, agarose gel electrophoresis, and the like.The accuracy of the standard function can be enhanced, as is known inthe art, by, e.g., obtaining standard data for increased numbers ofstandard concentrations, by testing each standard in higher numbers ofreplicates, by determining the standard quantities with higher accuracy,by using best fit regression analysis, and the like.

Standards and Test Samples

In vitro transcribed RNA is a preferred standard for many RNA analysesbecause large quantities of highly pure material can be obtained.Degraded IVT RNA can be obtained from full length undegraded IVT RNA byappropriate treatment. In preferred embodiments, the IVT RNA is degradedin the same fashion as the RNA to be analyzed, e.g., by age, light,chemicals, enzymes, heat, and/or the like. In many cases, fragmentationof the RNA is the type Of degradation with the most significant effecton an assay. This is particularly true for many RNA analyses based onhybridization reactions with the test RNA. In preferred embodiments ofthe invention, an IVT RNA standard material is degraded by exposure tohigh pH, resulting in fragmentation comparable to the known or expectedfragmentation of a test RNA sample.

It is envisioned that a standard function can be established for aquantitative RNA assay representing the relationship between assayoutput and sample input of RNA having various known degrees ofdegradation. Where the quantity of an RNA in a test sample is known,assay of the test sample with reference to the standard function canprovide a result indicating the degree of degradation for the test RNAsample.

The quantity of an RNA, such as a degraded RNA, can be determined basedon a quantitative assay and an standard function established usingdegraded RNA standards. The test sample RNA can be any type, such as,e.g., mRNA, rRNA, tRNA, IVT RNA, formalin (formaldehyde, methanol,water) treated RNA, RNA from dehydrated tissues, aged RNA (e.g., RNAfrom cells or tissue samples older than one year), RNA from humanclinical samples, FFPE cell and tissue samples, RNA samples frommicroscope slides, RNA samples degraded by exposure to RNase enzymes,and the like.

Assay Results

The quantity determined for a test RNA can be expressed, e.g., inrelative or absolute terms. The initial output value of an assay istypically in some unit of magnitude, such as, e.g., absorbance units,fluorescence units, relative light units (RLU), a voltage, a lightintensity, a radioactive particle count, and the like. The assay valuecan be input to a standard function to output something more tangible,such as a quantity of associated RNA, e.g., a mass, weight,concentration, number of moles, number of nucleotide bases, number ofmolecules, etc.

The choice of degraded NT RNA standard (or previously determinedstandard function) can be based on a known or expected degree ofdegradation in the test sample RNA. For example, based on experiencewith FFPE samples stored for different lengths of time (see, e.g., FIG.18 agarose gels), one could select, a standard with similar degradationfor preparation of a standard curve. Alternately, the degree ofdegradation of sample RNA could be evaluated for the actual test sampleby sizing techniques, such as, e.g., size exclusion chromatography, massspectroscopy, gel electrophoresis, by offset bDNA analysis (discussedbelow), and the like. Quantitative results for analysis of degraded. RNAcan often be improved by selecting standard functions based on knowledgeon hand.

Quantitative mRNA results determined using the degraded IVT RNA standardcurve can be input to further calculations. For example, where thenumber of cells is known that were the source of the mRNA, the amount ornumber of molecules of the mRNA per cell can be calculated. It isenvisioned that the number of cells represented by an RNA sample can beknown, e.g., by counting the cells, as discussed above, or can bedetermined, e.g., based on an rDNA assay with reference to anappropriate standard curve of cells versus rDNA.

Determining Nucleic Acid Degradation by Comparing Interspersed andOffset Probe Systems

A control bDNA assay for a nucleic acid with multiple capture extender(CE) complimentary target sequences dispersed along the nucleic acid,and multiple label extender (LE) complimentary target sequencesinterspersed along the nucleic acid, can generate a strong signal, evenif the nucleic acid is highly fragmented. In contrast, a test bDNA assaywith all or most of the CE target sequences at one end of the nucleicacid sequence and all or most of the LE target sequences spaced away atthe other end of the nucleic acid sequence can fail to generate asignal, e.g., if the tested nucleic acid is fragmented at a point in thespace between the CE and LE targets. This signal difference, dependenton the LE and CE target sequence locations can be utilized in assays toestimate the degree of fragmentation for a nucleic acid.

bDNA Assays

bDNA assays can generally be described as, e.g., capture of a targetnucleic acid by a capture extender associated with a solid support, thetarget being decorated at one or more point with label extenderstypically associated with branched DNA molecules capable of binding amultitude of label probes to generate a large signal.

For example, in one aspect of bDNA analysis, a target nucleic acid iscaptured and its presence on the solid support is detected using alabeled branched-chain DNA (bDNA-amplification multimer). Detecting thepresence of the target nucleic acid on the solid support can includehybridizing a first set of one or more label extenders (typically, twoor more label extenders) and a label probe system comprising a label tothe first target nucleic acid and detecting the presence of the label onthe solid support. The label probe system typically includes anamplification multimer and a plurality of label probes. Theamplification multimer is capable of hybridizing simultaneously to alabel extender and to a plurality of label probes. The label probe caninclude the label, or it can be configured to bind to the label.Suitable labels include, but are not limited to, an enzyme or afluorescent label. When an enzyme (e.g., alkaline phosphatase) is usedas the label, its activity can be detected with a chemiluminescent,colorimetric, or similar technique, as is well-known in the art. When afluorescent label is used, detecting the presence of the label on thesolid support typically comprises detecting a fluorescent signal fromthe label.

An exemplary embodiment in which a single: target nucleic acid iscaptured and detected using a bDNA assay is schematically illustrated inFIG. 17. A sample of cells or tissue is lysed to produce a lysateincluding target nucleic acid 114. The target nucleic acid 114 (e.g., anmRNA whose expression is to be detected) is captured by capture probe104 on solid support 101 (e.g., a well of a microtiter plate) throughset 111 of synthetic oligonucleotide capture extenders. Each captureextender has a first polynucleotide sequence C-3 (152) that canhybridize to the target nucleic acid and second polynucleotide sequenceC-1 (151) that can hybridize to the capture probe through sequence C-2(150) in the capture probe. Typically, two or more capture extenders areused; optionally, one CE can be used to capture a target. Each labelextender in label extenders set 121 hybridizes to a different sequenceon the target nucleic acid, through sequence L-1 (154) that iscomplementary to the target nucleic acid, and to sequence M-1 (157) onamplification multimer (141), through sequence L-2 (155). Blockingprobes (124), which hybridize to sequences in the target nucleic acidnot bound by either capture extenders or label extenders, are often usedin bDNA assays to reduce non-specific target probe binding. A probe setfor a given target nucleic acid thus consists of capture extenders,label extenders, and optional blocking probes 124 for the target nucleicacid. The capture extenders, label extenders, and optional blockingprobes are complementary to nonoverlapping sequences in the targetnucleic acid, and are typically, but not necessarily, contiguous. Inthis example, a single blocking probe is used; typically, an array ofdifferent blocking probes is used in an optimized bDNA assay.

Signal amplification can begin with the binding of the label extendersto the target nucleic acid. The amplification multimer is thenhybridized to the label extenders. The amplification multimer hasmultiple copies of sequence M-2 (158) that is complementary to labelprobe 142 (it is worth noting that the amplification multimer istypically, but not necessarily, a branched-chain nucleic acid; forexample, the amplification multimer can be a branched, forked, orcomb-like nucleic acid or a linear nucleic acid). Label 143, forexample, alkaline phosphatase, is covalently attached to each labelprobe. (Alternatively, the label can, e.g., be noncovalently associatedwith the label probes.) In the final step, labeled complexes aredetected, e.g., by the alkaline phosphatase-mediated degradation of achemilumigenic substrate, e.g., dioxetane. Luminescence is reported asrelative light units (RLUs) on a microplate reader. The amount ofchemiluminescence is proportional to the level of target nucleic acidoriginally present in the sample (a relationship describable with astandard function).

In the preceding example, the amplification multimer and the labelprobes comprise label probe system 140. In another example, the labelprobe system also comprises a preamplifier, e.g., as described in U.S.Pat. No. 5,635,352 and U.S. Pat. No. 5,681,697, which further amplifiesthe signal from a single target mRNA. In yet another example, the labelextenders hybridize directly to the label probes and no amplificationmultimer or preamplifier is used, so the signal from a single targetmRNA molecule is only amplified by the number of distinct labelextenders that hybridize to that mRNA.

Basic bDNA assays have been well described and have been used, e.g., todetect and quantify mRNA transcripts in cell lines and to determineviral loads. The bDNA assay provides direct quantification of nucleicacid molecules at physiological levels. Several advantages of thetechnology distinguish it from other DNA/RNA amplification technologies,including linear amplification, good sensitivity and dynamic range,great precision and accuracy, simple sample preparation procedure, andreduced sample-to-sample variation. For additional details on bDNAassays, see, e.g., U.S. Pat. No. 4,868,105 to Urdea et al. entitled“Solution phase nucleic acid sandwich assay”; U.S. Pat. No. 5,635,352 toUrdea et al. entitled “Solution phase nucleic acid sandwich assayshaving reduced background noise”; U.S. Pat. No. 5,681,697 to Urdea etal. entitled “Solution phase nucleic acid sandwich assays having reducedbackground noise and kits therefore”; U.S. Pat. No. 5,124,246 to Urdeaet al. entitled “Nucleic acid multimers and amplified nucleic acidhybridization assays using same”; U.S. Pat. No. 5,624,802 to Urdea etal. entitled “Nucleic acid multimers and amplified nucleic acidhybridization assays using same”; U.S. Pat. No. 5,849,481 to Urdea etal. entitled “Nucleic acid hybridization assays employing largecomb-type branched polynucleotides”; U.S. Pat. No. 5,710,264 to Urdea etal. entitled “Large comb type branched polynucleotides”; U.S. Pat. No.5,594,118 to Urdea and Horn entitled “Modified N-4 nucleotides for usein amplified nucleic acid hybridization assays”; U.S. Pat. No. 5,093,232to Urdea and Horn entitled “Nucleic acid probes”; U.S. Pat. No.4,910,300 to Urdea and Horn entitled “Method for making nucleic acidprobes”; U.S. Pat. No. 5,359,100; U.S. Pat. No. 5,571,670; U.S. Pat. No.5,614,362; U.S. Pat. No. 6,235,465; U.S. Pat. No. 5,712,383; U.S. Pat.No. 5,747,244; U.S. Pat. No. 6,232,462; U.S. Pat. No. 5,681,702; U.S.Pat. No. 5,780,610; U.S. Pat. No. 5,780,227 to Sheridan et al. entitled“Oligonucleotide probe conjugated to a purified hydrophilic alkalinephosphatase and uses thereof”; U.S. patent application Publication NoUS2002172950 by Kenny et al. entitled “Highly sensitive gene detectionand localization using in situ branched-DNA hybridization”; Wang et al.(1997) “Regulation of insulin preRNA splicing by glucose” Proc Nat AcadSci USA 94:4360-4365; Collins et al. (1998) “Branched DNA (bDNA)technology for direct quantification of nucleic acids: Design andperformance” in Gene Quantification, F Ferre, ed.; and Wilber and Urdea(1998) “Quantification of HCV RNA in clinical specimens by branched DNA(bDNA) technology” Methods in Molecular Medicine: Hepatitis C 19:71-78.In addition, reagents for performing basic bDNA assays (e.g.,QuantiGene™ kits, amplification multimers, alkaline phosphatase labeledlabel probes, chemilumigenic substrate, capture probes immobilized, on asolid support, and the like) are commercially available, e.g., fromPanomics, Inc. (on the world wide web at www.panomics.com), and can beadapted for the practice of the present invention. Software fordesigning probe sets for a given mRNA target (i.e., for designing theregions of the capture extenders, label extenders, and optional blockingprobes that are complementary to the target) is also commerciallyavailable (e.g., ProbeDesigner™ from Panomics, Inc.); see also Bushnellet al. (1999) “ProbeDesigner: for the design of probe sets for branchedDNA (bDNA) signal amplification assays Bioinforrnatics 15:348-55.

Offset Probe Assay

Variations on the bDNA assay can be used to detect fragmentation in atarget nucleic acid sequence. Fragmentation of the target nucleic acidcan be detected as a loss or reduced signal when a break exists in thenucleic acid sequence between separated target complimentary sequencesites for label extenders and capture extenders (which are bound to asolid support through capture probes). Such a break between the CEs andLEs can break the link between the labels and the solid support so thatlabel signals can be washed away in processing of the solid support. Thesensitivity and/or useful detection range of the assay can be improvedby comparison of the signal from an offset assay to a control assaywherein both CEs and LEs exist on both sides of the target nucleic acidbreak. A resulting ratio of offset to control signals can be moreindicative of fragmentation than absolute values, especially wheretarget nucleic acid quantities are unknown.

In an exemplary assay, as shown in FIGS. 18A and 18B, while the controlprobe set can provide a signal whether or not the target nucleic acid isfragmented, assay of the same fragmented target using the offset probesystem can fail to generate a signal. As shown in FIG. 18A, a controlprobe system of interspersed LE 121 and CE 111 probes can effectivelycapture full length target nucleic acid 114, which is decorated withdispersed label extenders 121, thus allowing generation of a strongcontrol signal. The full length target also allows a strong signal to begenerated in the offset probe system by connecting offset labelextenders to the solid support, through the capture extenders andcapture probes 104. As can be seen in FIG. 18B, signal continues to begenerated with the control probe set, even where the target nucleic acidsequence 177 in the test sample, is fragmented. The control probe setcaptures the target sequence (along with label extenders) on both sidesof the break so that the ability to generate a signal is not lost, e.g.,during process washes of the solid support. However, because the targetnucleic acid is broken in the space between offset system LE targetsequence sites and CE target sequence sites, no signal can be generatedusing the offset probe set of FIG. 18B. Although a fragment withoutbound LE probes is captured, the fragment with bound LE probes is not.Any signal associated with the bound offset LE probes will be lost whenhybridization solution is washed off the solid support. It is envisionedthat the positions of offset LEs and CEs can be established so that abreak (or lack thereof) at a particular region or point along a targetnucleic acid sequence can be detected.

In one embodiment, offset probes are designed so there is one CE targetsequence site at or near one end of the target nucleic and one or moreLE target sequence sites at the other end of the target nucleic acid. Inpreferred embodiments, the LE and CE sites are not immediately adjacent,but separated by a section of nucleic acid not complimentary to any LEor CE probe. A break anywhere between the CE complimentary target sitesand LE target sites would result in no signal generation represented forthat fragmented target by the offset system. The higher the proportionof target nucleic acids in a sample with such a break, the weaker thesignal that could be generated, thus enabling a quantitative standardcurve to be prepared using such a system. In a preferred embodiment, LEsites are present only near the two ends of the target nucleic acid,while one or more CE sites are present only between the two ends, e.g.,near the center of the target nucleic acid, e.g., separated by asequence not complimentary to any LEs or CEs of the system. In such acase, a break between the CE sites and one, end would reduce the abilityto generate a signal by, e.g., half; while breaks between the CE sitesand each end would eliminate the ability of the system to generate asignal altogether.

In preferred embodiments of the offset probe system, LE target sequencesites are designed for sensitivity to breakage between the site and thenearest 3′ and/or 5′ CE target sequence site on the target nucleic acid.For example, in many cases for this technology it is preferred thatthere be no CE site between the LE site and one end of the targetnucleic acid, so that if there is a break between the LE site and one ormore CE sites toward the other end, the LE will be lost (unassociatedwith a solid support) along with its signal potential. It is preferredthat a substantial portion of the target nucleic acid exist between theLE target sequences and CE target sequences. For example, in preferredembodiments, not less than 10% of the nucleic acid sequence of the testnucleic acid lies between the two closest LE and CE sites along thenucleic acid. It is more preferred that more than 25%, more 50%, morethan 75%, more than 90%, or more of the nucleic acid being tested forfragmentation be between the members of the closest CE/LE pair of sites.It is preferred that one or more offset label extender L1 sequences inthe offset probe system are complimentary to sequences of the nucleicacid spaced at least one nucleotide base either 5′ or 3′ from all the C3complimentary sequences; more preferably 5 or more, 10 or more, 25 ormore, 50 or more, 100 or more 500 or more bases are between the L1 andC3 complimentary sequences of the test nucleic acid.

In other embodiments of the offset probe scheme, there can be some CEtargets interspersed among LE targets on the target nucleic acid, and/orvisa versa. For example, an offset probe system can be designed whereinthe target nucleic acid is captured near the two ends while the LEtarget sequence sites are spaced away from the CE sites, e.g., near thecenter of the target nucleic acid. In such a case, the ability togenerate a signal would not be lost by a single break of the nucleicacid but could be lost with a break on each side of the LE target sites.It is envisioned that, additional variant configurations of CE/LEspacing and dispersion would gradually or ultimately result in the lossof an ability to generate a signal with enough breaks between CE and LEsites. However, in other preferred embodiments, not more than 25% of theLE target sites are between two or more CE target sites; or not morethan 25% of the CE sites are between two or more of the LE sites. Inmore preferred embodiments, not more than 10% of the LE target sites arebetween two or more CE target sites; or not more than 10% of the CEsites are between two or more of the LE sites. In more preferredembodiments, not more than 5% of the LE target sites are between two ormore CE target sites; or not more than 5% of the CE sites are betweentwo or more of the LE sites. In most preferred embodiments, CE and LEtarget sequence sites are not interspersed along the target nucleic acidsequence, i.e., no LE target sites are between any two CE target sitesand/or no CE sites are between any two LE sites.

To normalize the offset probe assay results and to aid in interassaycomparisons, it can be useful to provide fragmentation assay results inthe form of a ratio of test assay results to control assay results. Thecontrol assay, e.g., using multiple interspersed LE/CE probe targetsites, can be carried out on a different nucleic acid, the test nucleicacid full length, and/or with the test nucleic acid from the same samplebeing analyzed. In preferred embodiments the same test sample isanalyzed by both the control assay and offset assay to provide theratio. Particular ratios can be correlated to certain degrees offragmentation. For example, a standard curve of ratios versus degree offragmentation can be provided so that a ratio, for a particular samplecan be correlated to the level of fragmentation in that sample. Suchinformation can be useful, e.g., in a choice of degraded IVT RNAstandard to use in quantitation of an mRNA in the sample.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Comparison of Post-Homogenization Separation toPre-Homogenization Extraction of FFPE Tissue Slide HydrophobicComponents

To demonstrate equivalence and improvements of post-homogenizationseparation over pre-homogenization organic extraction, FFPE sample mRNAswere compared using the branched DNA technology with and withoutpre-solubilization dewaxing. Samples from >10 yr-old matched human lungnormal and tumor FFPE samples were pooled as three 10 micron sections.For one pair of samples (“O”), homogenates were prepared with waxseparation after solubilization. For a second pair of samples (“1”),homogenates were prepared with 1× dewaxing organic extraction stepbefore solubilization. For a third pair of samples (“2”), homogenateswere prepared with 2× dewaxing organic extraction cycles. Extracts weretested using ribosomal protein S3 (RPS3, NM_(—)001005 housekeeper orReference control RNA) and Lactate dehydrogenase A (LDHA, NM_(—)005566;2-3× induction in tumor samples) as described by Yang et al. 2006.Modified probe design software was developed to design oligonucleotideprobe sets for target genes in branch DNA assays (Bushnell et al.,1999). A probe set for a target gene consists of three types ofoligonucleotide probes (CE—capture extender, LE—label extender, andBL—blocking probe) covering a contiguous region of the target, whichallows the capture of target RNA to the surface of plate well andhybridization with branched DNA signal amplification molecule. For eachtarget sequence, the software algorithm identified regions that canserve as annealing templates for CEs (5-10 per gene), LEs (10-20 pergene), or BLs to substantially fill the remaining space. The branchedDNA assays were performed according to the procedure of QuantiGene®Reagent System (Panomics), which was previously described in detail(Wang et al., 1997, Kern et al., 1996). Briefly, 10 μL tissue homogenatewas mixed with 40 μL Lysis Mixture (Panomics), 40 μL Capture Buffer(Panomics), and 10 μl target gene-specific probe set (CE, 1.65 fmol/μL;LE, 6.6 fmol/μL; BL, 3.3 fmol/μL). Each sample mixture is then dispensedinto an individual well of a Capture Plate (Panomics).

As can be seen in the graphs of FIG. 3, the determination of mRNAexpression levels determined with post-solubilization separation iscomparable, if not improved, as compared to the 1× and 2× phaseextraction procedures.

Example 2 Equivalent Spike Recovery from FFPE Samples

An additional experiment was performed demonstrating that the recoveryof RNA from FFPE sections is equivalent for post-solubilizationseparation and pre-solubilization extraction. Methods were as describedabove for the Lactate dehydrogenase A (LDHA, NM_(—)005566) and ribosomalprotein S3 (RPS3, NM_(—)001005), except a known amount of in vitrotranscribed (IVT) RNA from the bacterial gene dihydrodipicolinatereductase (dapB, L38424 not expressed in the human lung tissue) wasadded to a solubilized FFPE sample. The data show yields were comparablebetween the 2× dewax extraction technique and physical wax separationtechnique. Using either procedure, the capture efficiency (spikerecovery) of the spiked-in dapB IVT RNA ranged from 90-110%.

Example 3 Use of Repetitive Ribosomal DNA to Determine Efficiency ofFFPE Tissue Solubilization and Number of Cells in the FFPE Sample

An investigation was Made to determine if accurate estimates of cellnumbers can be made based on repetitive DNAs. In particular, cell countsfor tumor and other aneuploid cells were determined using standardcurves based on quantitation of ribosomal DNA genes.

Ten (10) cell lines were chosen with an average of about ten (10)ribosomal gene clusters per diploid genome. Four thousand (4000) cellsof each cell line were lysed in QuantiGene Lysis Buffer (Panomics) and10 cell line lysates were pooled; i.e. 10 cell lines in total or 40,000cells are in a total volume of 110 ul.

Thirty microliters (30 ul) each were transferred to separate microfugetubes, one to denature the DNA to measure the ribosomal DNAs (18S & 28SrDNA) and another undenatured control to measure background of theassay. Next, each tube was diluted to 300 ul total by adding 270 μl TEbuffer. 180 ul each of denature and control samples were transferred toreaction tubes. 18 ul of 2.5N.NaOH was added to the tube containing theDNA for denaturing and 18 ul TE was added to the control aliquot beforeheating the tubes at 53° C. for 15 minutes (see FIG. 6). After theheating step, 90 ul of 2M HEPES was added to both control (undenatured)and denatured samples. The DNA in the control sample remained doublestranded, whereas the denatured DNA sample was substantially in thesingle-stranded form. Cell number standard curves were, established byadding denatured samples (30 ul, 15 ul, 7.5 ul, 3.8 ul, 1.9 ul, 0.95 ulor 683, 341, 171, 85, 43, 21 cell equivalents, respectively) toQuantiGene assays (see FIG. 7). Instead of using typical antisense probesets to measure mRNA, sense probe sets are used to quantify the amountof 185 and 28S ribosomal DNA in the samples.

Because the 18S and 28S ribosomal probe sets gave virtually the sameresults for the pool of cell lines, the amount of ribosomal DNAs in FFPEtumor samples and FFPE cell line controls was determined using only the18S ribosomal probe sets. FFPE Tumor and cell line control sections weresolubilized and tested as described above. Briefly, sections weresolubilized in 300 ul QuantiGene Homogenization Buffer and 2 ul diluted10³. 60 ul of 10³ diluted sample was transferred to a microfuge tube anddenatured by adding 6 ul of 2.5N NaOH followed by heating to 53° C. for15 minutes. At the end of denaturation the solution was neutralized byadding 30 ul of 2M HEPES with a vortex mix. 30 ul of the denaturedsolution was added to the QuantiGene assay and 18S ribosomal DNA wasquantified using the 18S rDNA cell number standard curve (pooled 10 celllines) as described above.

A known number of cells from four cell lines were fixed with formalinand embedded in paraffin. Twenty sections (6 um×20 mm²=˜100,000cells/section=total˜2×10⁶ cells) of each cell line were solubilized in600 ul QuantiGene Homogenization Buffer, DNA denatured, and quantifiedusing the 18S rDNA probe sets as described in above. Using the 18S rDNAcell number standard curve, cell counts between 2-2.5×10⁶ weredetermined for the cell line FFPE samples each with 20 sections. SeeFIG. 8.

Example 4 Cell Number Quantitation from FFPE Tumor Tissue Sections

Ten sections (6 um×24 mm²; all sections>85% tumor) each from 49 tumors(24 lung, 25 colon) were solubilized in 300 ul QuantiGene HomogenizationBuffer, DNA denatured, and quantified using the 18S rDNA probe sets asdescribed in above. Using the 18S rDNA cell number standard curve,between 1.2-1.7×10⁶ cells/FFPE tumor sample were quantified for 10sections total. See FIG. 9.

Example 5 Quantifying RNA Molecules Using Intact and Degraded IVT RNAStandard Curves

In Vitro Transcribed RNAs (IVTs) using cloned genes can be used asstandard curves to calculate the number of RNA molecules. FFPE mRNAs aretypically somewhat degraded, e.g., by fragmentation processes. We havefound that degraded IVT standard analysis curves can be compared tocurves for undegraded full length RNAs to determine an assay efficiencyfor analysis of a degraded sample. For example, the QuantiGene assay canbe used to accurately quantify degraded mRNA from clinical samples.

IVTs were synthesized according to Ambion instructions. A portion of theintact. RNAs were degraded to 100-300 bp to mimic the size of FFPE RNAs.To accomplish the desired size of degraded RNAs, undegraded full lengthIVT RNAs are degraded in 0.1N NaOH for 9 minutes. The reaction wasneutralized using an equivolume of 0.1N HCl to stop the degradationprocess. The gel shown in FIG. 10 shows seven undegraded full length invitro transcribed RNAs (pooled in lane 2) and the same seven IVT RNAsdegraded and pooled for the gel electrophoresis.

Next the IVT concentration was determined and the solutions seriallydiluted to 40, 10, 2.5, 0.625, 0.156, and 0.039 attamole (⁻¹⁸ mol). Asshown in FIG. 11, for example, beta-Actin (ACTB) IVT undegraded fulllength RNA (1739 bp) and corresponding IVT degraded RNA (see gel FIG.10) were quantified using beta-Actin specific probe sets (Panomics) andstandard curves established. When the standard curves are compared toeach other, a R² value of 1 is determined (R-squared refers to thefraction of variance). As can be seen; the sensitivity of the ACTBspecific probe sets using degraded ACTB IVT (100-300 bp) is ˜40% (slope38.6%) of the undegraded ACTB IVT (1739 bp) for all concentrations.Similar findings were found for the other 6 genes using both undegradedfull length (ranging in size from 985 bp to 4407 bp) and degraded IVTs(see FIG. 12).

Known numbers of cells for 4 cell lines (previously discussed at the endof Example 3) were fixed with formalin and embedded in paraffin (twentysections, 6 um×20 mm²=˜100,000 cells/section=total˜2×10⁶ cells) andsolubilized in 600 ul QuantiGene Homogenization Buffer were used toquantify the amount using equivolumes of input samples for all 6 mRNAsin all 4 cell lines. First, bDNA assays were used to determine relativeamounts of expression in each cell line for each of 6 mRNAs (see FIG.13). Then, from the raw data, the attomoles of each of the 6 mRNAs wascalculated using both IVT standard curves (undegraded & degraded, seeabove). Finally, the attomoles were divided by the cell number of eachcell line using the 18S rDNA/cell number standard curve to determinecopy number per cell for each mRNA and cell line (see. FIG. 14). Thecopy number calculated from the degraded IVT standard curves correlatedclosely with the copy numbers calculated for the fresh full length IVTRNA samples using the undegraded IVT standard curve. Thus, the degradedIVTs better represent mRNAs derived from homogenized FFPE samples, andprovide better standard materials for quantitation of such mRNAs.

In an additional example of mRNA quantitation and copy numberdetermination, the ten sections (6 um×24 mm²; all sections>85% tumor)each from 49 tumors (25 lung, 24 colon) were solubilized in 300 ulQuantiGene Homogenization Buffer as described above for the 185 and 28SrDNAs. The resultant homogenates were used to quantitate the amount ofthe 6 mRNAs using the QuantiGene specific probe sets (see above). IVTstandard curves were the same used to measure the mRNAs in the 4 celllines. FIG. 15 shows the raw relative, quantitative data for each of themRNAs in all 49 tumors.

The attomoles of each mRNA in all tumors was calculated from thedegraded IVT standard curve and divided by the number of cells in eachof the tumor samples (total of 10 sections) for use in calculating themRNA copies per cell. This data is shown in FIG. 16. It was noted that aclear trend exists with the copy number per cell for expression levels(mRNAs) in some of the genes being elevated in the lung tumors (1-25)and not in the colon tumors (26-49).

Example 6 Determination of Sample RNA Degradation

Fragment length of an RNA was assessed for a tissue block. Thisassessment can aid in, e.g., selection of FFPE blocks with adequate RNAintegrity and in choice of an IVT RNA assay standard for use with asample. After FFPE solubilization, a sample was tested using two probesets for an mRNA (gene) of interest. Either standard dispersed controlprobe sets (see FIGS. 18A and 18B) or test probe sets with off-set CEand LE sites were are added to the tissue homogenizing buffer for overnight hybridization. The control and test samples were processed thenext day according to standard bDNA assay procedures.

As can be seen in FIG. 19, FFPE samples of varying RNA quality weretested. First the quality of purified RNA is evaluated by gelelectrophoresis. The RNA in 3 year old samples (both tumor and normalRNA) presented less fragmentation than those of the year old samples.Next, intact control RNA and RNA from both 3 year old and 10 year oldFFPE samples were subjected to bDNA analysis with either dispersedcontrol probes or off-set fragmentation test probes. The quality of theRNA was assessed by determining the ratio of the assay signal forcontrol over offset assay results. We found the ratio increases withincreasing RNA degradation (control ratio=1; 3 year old=˜3-4; 10 yearold=7-24). The offset bDNA probe scheme offers a simple approach toassess the quality of RNA from any sample.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, many of the techniques and apparatus describedabove can be used in various combinations.

All publications, patents, patent applications, and/or other documentscited in this application are incorporated by reference in theirentirety for all purposes to the same extent as if each individualpublication, patent, patent application, and/or other document wereindividually indicated to be incorporated by reference for all purposes.

1-19. (canceled)
 20. A method of determining a number of test cells, themethod comprising: obtaining a reference nucleic acid sample from aknown number of reference cells; quantitating an amount of a ribosomalDNA in the reference sample; providing a standard function for thereference cell number versus the reference ribosomal DNA quantity;obtaining a test nucleic acid sample from test cells; quantitating anamount of the ribosomal DNA in the test sample; and, determining a testcell number based on the standard function and the quantity of testribosomal DNA.
 21. The method of claim 20, wherein the test cell isselected from the group consisting of: a tumor cell, a cell line, cellson a microscope slide, FFPE cells, and a polyploid cell.
 22. The methodof claim 21, wherein the tumor cell comprises a lung tumor cell or acolon tumor cell.
 23. The method of claim 20, wherein said obtaining thereference nucleic acid sample comprises nucleic acid extraction fromcells having a substantially normal karyotype.
 24. The method of claim20, wherein the ribosomal DNA is selected from the group consisting of:18S rDNA, 5.8S rDNA and 28S rDNA.
 25. The method of claim 20, whereinsaid quantitating comprises a technique selected from the groupconsisting of: bDNA analysis; Southern blot analysis, polymerase chainreaction, and agarose gel electrophoresis.
 26. The method of claim 20,wherein said determining the cell number comprises a method selectedfrom the group consisting of: inputting the test ribosomal DNA quantityinto the standard function, inputting the test ribosomal DNA quantityinto a formula comprising a ratio of cells to rDNA, and inputting thetest rDNA value into a computer.
 27. The method of claim 20, furthercomprising normalizing a result of a test cell analysis using thedetermined test cell number.
 28. The method of claim 20, furthercomprising determining an efficiency of test nucleic acid extractionbased on a known test cell number and the determined test cell number.29. A method of determining RNA copy numbers, the method comprising:determining a number of cells in a test sample; providing a standardfunction for an RNA assay output versus a degraded in vitro RNA standardassay input; determining an amount of a test RNA in the test sample bythe RNA assay using the standard function; and, determining the copynumber of the RNA in the cells based on the number of cells and thedetermined amount of test RNA.
 30. The method of claim 29, wherein thetest sample is selected from the group consisting of: a tumor cell, acell line, cells from a microscope slide, clinical samples more than ayear old, cells fixed with formalin, cells embedded in paraffin, and aaneuploid cell.
 31. The method of claim 29, wherein the RNA standard isdegraded by a degradation source selected from the group consisting of:time of storage, high pH, low pH, shear stress, a nuclease, light, heat,and contact with formaldehyde.
 32. The method of claim 29, wherein thequantitative analysis comprises a method selected from the groupconsisting of: bDNA analysis, northern blot analysis, RT-polymerasechain reaction, spectrophotometry, fluorometry, and agarose gelelectrophoresis.
 33. A method of determining a RNA copy number for cellsin a FFPE sample, said method comprising: providing a standard functionfor cell number versus quantity of a repetitive DNA; separating paraffinfrom the cells after incubation in a protease solution at 40° C. ormore; determining a number of cells represented in the protease solutionbased on the standard function and an amount of the repetitive DNA inthe protease solution; providing a standard function of RNA assay outputversus a degraded IVT RNA standard assay input; determining an amount ofthe RNA in the protease solution based on the degraded IVT RNA standardfunction; and, calculating the RNA copy number per cell by dividing thedetermined number of cells by the determined amount of RNA.
 34. Themethod of claim 33, wherein the repetitive DNA comprises a ribosomalDNA.
 35. The method of claim 33, wherein the protease is a proteinase Kand the incubation temperature is about 65° C.
 36. The method of claim33, wherein the RNA assay comprises a bDNA assay, RT-PCR, or a northernblot.
 37. The method of claim 33, further comprising selecting thedegraded IVT RNA standard or modifying a slope of a standard curveaccording to the results of an offset bDNA assay.
 38. A method ofdetecting fragmentation of a nucleic acid, the method comprising:analyzing a sample of the nucleic acid in an offset bDNA assay whereineach of one or more offset capture extender probe C3 sequences arecomplimentary to sequences along the nucleic acid, and wherein one ormore offset label extender L1 sequences are complimentary to sequencesof the nucleic acid spaced at least one nucleotide base either 5′ or 3′from all the C3 complimentary sequences; wherein less signal isgenerated in the bDNA assay if the nucleic acid is fragmented betweenthe C3 and L1 complimentary sequences than if the nucleic acid is notfragmented between the C3 and L1 complimentary sequences.
 39. The methodof claim 38, further comprising: analyzing the sample of the nucleicacid in a second bDNA assay wherein two or more control capture extenderprobe C3 sequences are complimentary to sequences at different positionsalong the nucleic acid, and wherein one or more control label extenderL1 sequences are complimentary to sequences at different positions alongthe nucleic acid sequence; with one or more of the control L1 sequencesbeing complimentary to the nucleic acid at positions between thepositions complimentary to two or more of the control C3 sequences;wherein, if the nucleic acid is fragmented, a ratio of a control assayresult over the offset assay result is higher than the ratio if thenucleic acid is not fragmented.
 40. The method of claim 38, wherein thenucleic acid is fragmented.
 41. The method of claim 38, wherein thenucleic acid is selected from the group consisting of: an RNA, an mRNA,a cDNA, a nucleic acid from a FFPE sample, a nucleic acid that does nothave intron sequences, and a nucleic acid from a cell or tissue samplemore than a year old.
 42. The method of claim 38, wherein the nucleicacid sequences complimentary to the offset C3 sequences are separatedfrom the nucleic acid sequences complimentary to the offset L1 sequencesby a space of 75% or more of the nucleic acid nucleotides.
 43. Themethod of claim 38, wherein the one or more offset label extender L1sequences are complimentary to sequences of the nucleic acid spaced atleast 25 nucleotide bases either 5′ or 3′ from all the C3 complimentarysequences.
 44. The method of claim 38, wherein the one or more spacingnucleotides comprise a sequence complimentary to a blocking probe. 45.The method of claim 38, wherein no L1 complimentary sequence is betweenany two C3 complimentary sequences.
 46. The method of claim 39, furthercomprising correlating the ratio to an average fragment length of thenucleic acid.
 47. The method of claim 39, further comprising selectingan assay standard or selecting a standard function based on the ratio.